Vishal Nigam

Background: The use of cardiopulmonary bypass (CPB) can induce sterile systemic inflammation that contributes to morbidity and mortality, especially in children. Patients have been found to have increased expression of cytokines and transmigration of leukocytes during and after CPB. Previous work has demonstrated that the supraphysiologic shear stresses present during CPB are sufficient to induce proinflammatory behavior in non-adherent monocytes. The interactions between shear stimulated monocytes and vascular endothelial cells have not been well studied and have important translational implications. Methods: To test the hypothesis that non-physiological shear stress experienced by monocytes during CPB affects the integrity and function of the endothelial monolayer via IL-8 signaling pathway, we have used an in vitro CPB model to study the interaction between THP-1 monocyte-like cells and human neonatal dermal microvascular endothelial cells (HNDMVECs). THP-1 cells were sheared in polyvinyl chloride (PVC) tubing at 2.1 Pa, twice of physiological shear stress, for 2 hours. Interactions between THP-1 cells and HNDMVECs were characterized after coculture. Results: We found that sheared THP-1 cells adhered to and transmigrated through the HNDMVEC monolayer more readily than static controls. When co-culturing, sheared THP-1 cells also disrupted in the VE-cadherin and led to reorganization of cytoskeletal F-actin of HNDMVECs. Treating HNDMVECs with IL-8 resulted in upregulation of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) while also increasing the adherence of non-sheared THP-1 cells. Preincubating HNDMVECs with Reparixin, an inhibitor of CXCR2/IL-8 receptor inhibited sheared THP-1 cell adhesion to the HNDMVECs. Conclusions: These results suggested that IL-8 not only increases the endothelium permeability during monocyte migration, but also affects the initial adhesion of monocytes in a CPB setup. This study revealed a novel mechanism of post-CPB inflammation and will contribute to the development of targeted therapeutics to prevent and repair the damage to neonatal patients. Highlights: Shear stress in a CPB-like environment promoted the adhesion and transmigration of monocytes to and through endothelial monolayer. Treating endothelial monolayer with sheared monocytes led to disruption of VE-cadherin and reorganization of F-actin. Interaction between sheared monocytes resulted in a significant increase of IL-8 release. Inhibiting IL-8 receptor prevented sheared monocyte adhesion, while IL-8 promoted naive monocyte adhesion.

Background: The systemic inflammation that occurs after exposure to cardiopulmonary bypass (CPB), which is especially severe in neonatal patients, is associated with poorer outcomes and is not well understood. In order to gain deeper insight into how exposure to bypass activates inflammatory responses in circulating leukocytes, we studied changes in microRNA (miRNA) expression during and after exposure to bypass. miRNAs are small noncoding RNAs that have important roles in modulating protein levels and function of cells. Methods and Results: We performed miRNA‐sequencing on leukocytes isolated from neonatal patients with CPB (n=5) at 7 time points during the process of CPB, including before the initiation of bypass, during bypass, and at 3 time points during the first 24 hours after weaning from bypass. We identified significant differentially expressed miRNAs using generalized linear regression models, and miRNAs were defined as statistically significant using a false discovery rate–adjusted P <0.05. We identified gene targets of these miRNAs using the TargetScan database and identified significantly enriched biological pathways for these gene targets. We identified 54 miRNAs with differential expression during and after CPB. These miRNAs clustered into 3 groups, including miRNAs that were increased during and after CPB (3 miRNAs), miRNAs that decreased during and after CPB (10 miRNAs), and miRNAs that decreased during CPB but then increased 8 to 24 hours after CPB. A total of 38.9% of the target genes of these miRNAs were significantly differentially expressed in our previous study. miRNAs with altered expression levels are predicted to significantly modulate pathways related to inflammation and signal transduction. Conclusions: The unbiased profiling of the miRNA changes that occur in the circulating leukocytes of patients with bypass provides deeper insight into the mechanisms that underpin the systemic inflammatory response that occurs in patients after exposure to CPB. These data will help the development of novel treatments and biomarkers for bypass‐associated inflammation.

Patients undergoing cardiac surgery face significant inflammatory induced by exposure to cardiopulmonary bypass (CPB), contributing to heightened morbidity and mortality. The molecular and cellular mechanisms that underpin this inflammatory process remain unknown. To address this knowledge gap, we performed snRNA/ATAC-Sequencing on leukocytes from neonatal CPB patients. Classical monocytes become more prevalent and have dysregulation of inflammatory genes after CPB, indicating their role in CPB-associated inflammation. A genome-wide CRISPR screen and in vitro experiments in non-adherent monocytic cells identified two novel genes, SPTAN1 and RAF1, as effectors of hemodynamic stress. SPTAN1 and RAF1 activate store-operated calcium entry that results inflammation and cell death. snATAC-Seq revealed dynamically changing patterns of chromatin accessibility and AP-1 transcription factor binding after CPB exposure. These findings provide novel insights into the pathogenesis of CPB-associated inflammation, with broad implications for understanding the early stages of sterile inflammation and how non-adherent cells sense shear stress.

Objectives The systemic inflammation that occurs after exposure to cardiopulmonary bypass (CPB), which is especially severe in neonatal patients, is associated with poorer outcomes and is not well understood. In order to gain deeper insight into how exposure to bypass activates inflammatory responses in circulating leukocytes, we studied changes in microRNA (miRNA) expression during and after exposure to bypass. miRNAs are small non-coding RNAs that have important roles in modulating protein levels and function of cells. Methods We performed miRNA-Sequencing on leukocytes isolated from neonatal cardiopulmonary bypass patients (N=5) at 7 timepoints during the process of CPB, including prior to the initiation of bypass, during bypass, and at three time points during the first 24 hours after weaning from bypass. We identified significant differentially expressed miRNAs using generalized linear regression models, and miRNAs were defined as statistically significant using an FDR adjusted p Results We identified 54 miRNAs with differential expression during and after CPB. These miRNAs clustered into 3 groups, including miRNAs that were increased during and after CPB (3 miRNAs), miRNAs that decreased during and after CPB (10 miRNAs), and miRNAs that decreased during CPB but then increased 8-24 hours after CPB. 38.9% of the target genes of these miRNAs were significantly differentially expressed in our previous study. miRNAs with altered expression levels are predicted to significantly modulate pathways related to inflammation and signal transduction. Conclusions The unbiased profiling of the miRNA changes that occur in the circulating leukocytes of bypass patients provides deeper insight into the mechanisms that underpin the systemic inflammatory response that occurs in patients after exposure to cardiopulmonary bypass. These data will help the development of novel treatments and biomarkers for bypass associated inflammation.

Dysregulated protein degradative pathways are increasingly recognized as mediators of human disease. This mechanism may have particular relevance to desmosomal proteins that play critical structural roles in both tissue architecture and cell-cell communication, as destabilization/breakdown of the desmosomal proteome is a hallmark of genetic-based desmosomal-targeted diseases, such as the cardiac disease arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C). However, no information exists on whether there are resident proteins that regulate desmosomal proteome homeostasis. Here, we uncovered a cardiac constitutive photomorphogenesis 9 (COP9) desmosomal resident protein complex, composed of subunit 6 of the COP9 signalosome (CSN6), that enzymatically restricted neddylation and targeted desmosomal proteome degradation. CSN6 binding, localization, levels, and function were affected in hearts of classic mouse and human models of ARVD/C affected by desmosomal loss and mutations, respectively. Loss of desmosomal proteome degradation control due to junctional reduction/loss of CSN6 and human desmosomal mutations destabilizing junctional CSN6 were also sufficient to trigger ARVD/C in mice. We identified a desmosomal resident regulatory complex that restricted desmosomal proteome degradation and disease.

Cardiopulmonary bypass (CPB) is required during most cardiac surgeries. CBP drives systemic inflammation and multiorgan dysfunction that is especially severe in neonatal patients. Limited understanding of molecular mechanisms underlying CPB-associated inflammation presents a significant barrier to improve clinical outcomes. To better understand these clinical issues, we performed mRNA sequencing on total circulating leukocytes from neonatal patients undergoing CPB. Our data identify myeloid cells, particularly monocytes, as the major cell type driving transcriptional responses to CPB. Furthermore, IL-8 and TNF-α were inflammatory cytokines robustly upregulated in leukocytes from both patients and piglets exposed to CPB. To delineate the molecular mechanism, we exposed THP-1 human monocytic cells to CPB-like conditions, including artificial surfaces, high shear stress, and cooling/rewarming. Shear stress was found to drive cytokine upregulation via calcium-dependent signaling pathways. We also observed that a subpopulation of THP-1 cells died via TNF-α–mediated necroptosis, which we hypothesize contributes to post-CPB inflammation. Our study identifies a shear stress–modulated molecular mechanism that drives systemic inflammation in pediatric CPB patients. These are also the first data to our knowledge to demonstrate that shear stress causes necroptosis. Finally, we observe that calcium and TNF-α signaling are potentially novel targets to ameliorate post-CPB inflammation.

Perturbations in biomechanical stimuli during cardiac development contribute to congenital cardiac defects such as hypoplastic left heart syndrome (HLHS). This study sought to identify stretch-responsive pathways involved in cardiac development. miRNA-Seq identified miR-486 as being increased in cardiomyocytes exposed to cyclic stretch in vitro. The right ventricles (RVs) of patients with HLHS experienced increased stretch and had a trend toward higher miR-486 levels. Sheep RVs dilated from excessive pulmonary blood flow had 60% more miR-486 compared with control RVs. The left ventricles of newborn mice treated with miR-486 mimic were 16.9%–24.6% larger and displayed a 2.48-fold increase in cardiomyocyte proliferation. miR-486 treatment decreased FoxO1 and Smad signaling while increasing the protein levels of Stat1. Stat1 associated with Gata-4 and serum response factor (Srf), 2 key cardiac transcription factors with protein levels that increase in response to miR-486. This is the first report to our knowledge of a stretch-responsive miRNA that increases the growth of the ventricle in vivo.

Introduction: Hypoplastic left heart syndrome (HLHS) is a congenital condition with an under-developed left ventricle (LV) that provides inadequate systemic blood flow postnatally. The development of HLHS is postulated to be due to altered biomechanical stimuli during gestation. Predicting LV size at birth using mid-gestation fetal echocardiography is a clinical challenge critical to prognostic counseling. Hypothesis: We hypothesized that decreased ventricular filling in utero due to mitral stenosis may reduce LV growth in the fetal heart via mechanical growth signaling. Methods: We developed a novel finite element (FE) model of the human fetal heart in which cardiac myocyte growth rates are a function of fiber and cross-fiber strains, which is affected by altered ventricular filling, to simulate alterations in LV growth and remodeling. Model results were tested with echocardiogram measurements from normal and HLHS fetal hearts. Results: A strain-based fetal growth model with a normal 22 week ventricular filling (1.04 ml) was able to replicate published measurements of changes between mid-gestation to birth of mean LV end diastolic volume (EDV) (1.1 to 8.3 ml) and dimensions (long-axis 18 to 35mm; short-axis 9 to 18mm) within 15% RMSD error. By decreasing volumetric load (-25%) at mid-gestation in the model, which emulates mitral stenosis in utero, a 65% reduction in LV EDV and a 46% reduction in LV wall volume were predicted at birth, similar to observations in HLHS patients. In retrospective blinded case studies for HLHS, using mid-gestation echocardiographic data, the model predicted a borderline and severe hypoplastic LV, consistent with the patients’ late-gestation data in both cases. Notably the model prediction was validated by testing for changes in LV shape in the model against clinical data for each HLHS case study. Conclusion: Reduced ventricular filling and altered shape may lead to reduced LV growth and a hypoplastic phenotype by reducing myocardial strains that serve as a myocyte growth stimulus. The human fetal growth model presented here may lead to a clinical tool that can help predict LV size and shape at birth based on mid-gestation LV echocardiographic measurements.